Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption

Abstract

The gene (1,542 bp) encoding thermostable Ca(2+)-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t 1/2 of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir-Hinshelwood kinetic analysis in terms of equilibrium parameter (K R) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t 1/2 of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs.

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

“…5 to 7 (1). The functions of CBM25 domains have been described previously, and their three-dimensional (3D) structures have been resolved (30,(44)(45)(46). It has also been demonstrated that the CBM25-26 tandem in the maltohexaose-forming amylase from Bacillus halodurans C-125 has a 50-fold stronger binding affinity for granular corn starch than each of the single domains (44).…”

Raw starch degrading α-amylases: an unsolved riddle

“…This observation leads to the idea that Trp201 and Trp202 of BaqA might represent a fingerprint of the new GH13 α-amylase subfamily [69]. These two tryptophans are also conserved in Geobacillus thermoleovorans α-amylases GTA [73] and Gt-amyII [61] as W204 and W205 with an important difference that in these two cases W205 is not located on the surface of the protein but hidden within the structure. On the other hand, the role of domain C in raw starch adsorption has been proven by truncation and LangmuirHinshelwood adsorption experiments [61].…”

“…There are not many α-amylases that are able to perform effectively on high concentration of raw starch (>25%) [28, [60][61][62][63][64][65] (Table 1), due to substrate or product inhibition [66] and because of the need for higher enzyme concentrations in raw starch digestion, which have to be obtained through the optimized upstream and downstream processing to obtain high quantity of biocatalyst. It is likely that during amylase attack on starch granule, enzymestarch physical, irreversible or non-catalytic binding may be both, productive and non-productive, which is why high enzyme loadings give rise to the formation of productive interactions [8].…”

“…These two tryptophans are also conserved in Geobacillus thermoleovorans α-amylases GTA [73] and Gt-amyII [61] as W204 and W205 with an important difference that in these two cases W205 is not located on the surface of the protein but hidden within the structure. On the other hand, the role of domain C in raw starch adsorption has been proven by truncation and LangmuirHinshelwood adsorption experiments [61]. These findings lead recently to an establishment of a novel subfamily of GH family GH13 for α-amylases from Anoxybacillus species (ASKA and ADTA) [74], G. thermoleovorans (GTA [73], Pizzo [75], Gt-amyII [61]), and B. aquimaris (BaqA) [69].…”

“…On the other hand, the role of domain C in raw starch adsorption has been proven by truncation and LangmuirHinshelwood adsorption experiments [61]. These findings lead recently to an establishment of a novel subfamily of GH family GH13 for α-amylases from Anoxybacillus species (ASKA and ADTA) [74], G. thermoleovorans (GTA [73], Pizzo [75], Gt-amyII [61]), and B. aquimaris (BaqA) [69]. Members of this new GH13 subfamily act on raw starch and are characterized by a C-terminus composed of five conserved aromatic residues, two tryptophan residues between CSR-V and CSR-II, and an LPDlx motif in CSR-V [76].…”